Kuypers L C, Decraemer W F, Dirckx J J J, Timmermans J-P
Laboratory of Biomedical Physics, Department of Physics, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerpen, Belgium.
J Microsc. 2005 Apr;218(Pt 1):68-78. doi: 10.1111/j.1365-2818.2005.01457.x.
A difference in refractive index (n) between immersion medium and specimen results in increasing loss of intensity and resolution with increasing focal depth and in an incorrect axial scaling in images of a confocal microscope. Axial thickness measurements of an object on such images are therefore not exact. The present paper describes a simple procedure to determine the correct axial thickness of an object with confocal fluorescence microscopy. We study this procedure for a specimen that has a higher refractive index than the immersion medium and with a thickness up to 100 microm. The measuring method was experimentally tested by comparing the thickness of polymer layers measured on axial images of a confocal microscope in case of a water-polymer mismatch to reference values obtained from an independent technique, i.e. scanning electron microscopy. The case when the specimen has a lower refractive index than the immersion medium is also shown by way of illustration. Measured thickness data of a water layer and an oil layer with the same actual thickness were obtained using an oil-immersion objective lens with confocal microscopy. Good agreement between theory and experiment was found in both cases, consolidating our method.
浸液介质与样本之间的折射率（n）差异会导致随着焦深增加，强度和分辨率损失增大，并且共聚焦显微镜图像中的轴向缩放比例不正确。因此，在此类图像上对物体进行的轴向厚度测量并不准确。本文描述了一种使用共聚焦荧光显微镜确定物体正确轴向厚度的简单方法。我们针对折射率高于浸液介质且厚度达100微米的样本研究了该方法。通过比较共聚焦显微镜轴向图像上测量的聚合物层厚度（在水与聚合物不匹配的情况下）与通过独立技术（即扫描电子显微镜）获得的参考值，对测量方法进行了实验测试。还通过示例展示了样本折射率低于浸液介质的情况。使用油浸物镜通过共聚焦显微镜获得了具有相同实际厚度的水层和油层的测量厚度数据。在这两种情况下均发现理论与实验结果吻合良好，从而证实了我们的方法。
